Glaesserella parasuis QseBC two-component system senses epinephrine and regulates capD expression

ABSTRACT Glaesserella parasuis, a common inhabitant of the upper respiratory tract of pigs, is the etiological agent of Glässer’s disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis. Decreased production of capsular polysaccharide (CPS) promotes bacterial adhesion and uptake, whereas maximal expression of CPS is essential to evade the host immune response. For bacterial survival under highly variable host conditions, a coordinated regulation of CPS expression is necessary. In the present study, we investigated how epinephrine (Epi) affects the CPS production, biofilm formation, and adhesion ability of G. parasuis, as well as the specific mechanism of G. parasuis changes in response to Epi. The results demonstrated that Epi stimulation dramatically inhibited CPS expression, and both the action of Epi and the deletion of CPS biosynthesis gene capD had detrimental impacts on biofilm formation, and had the enhanced ability for cell adhesion. QseBC two-component system is a typical adrenaline-sensing system in bacteria. In the present study, by quantitative real-time PCR, Western blot, and electrophoretic mobility shift assay, we discovered that in the presence of Epi, G. parasuis negatively regulated capD expression via the interaction of QseB with the promoter of capD, thereby affecting the synthesis of CPS. In conclusion, we explored the impact of Epi on CPS synthesis and defined its regulatory mechanism in G. parasuis for the first time, which provides an important theoretical basis for understanding the pathogenic mechanism of G. parasuis. IMPORTANCE The key bacterial pathogen Glaesserella parasuis, which can cause Glässer’s disease, has caused significant financial losses to the swine industry worldwide. Capsular polysaccharide (CPS) is an important virulence factor for bacteria, providing the ability to avoid recognition and killing by the host immune system. Exploring the alteration of CPS synthesis in G. parasuis in response to epinephrine stimulation can lay the groundwork for revealing the pathogenic mechanism of G. parasuis as well as providing ideas for Glässer’s disease control.

the amount of capsular material might significantly enhance adherence and uptake (7), whereas it may also convert the bacteria to become stronger in terms of their ability to elude the host immune system (8).As a result, for a pathogen to colonize, survive, and disseminate within the host, both the conversion from highly encapsulated to less encapsulated bacteria and the retrograde conversion must be carefully regulated (9).However, the mechanism through which bacteria regulate the production of CPS remains largely unknown.The capD gene, which encodes a polysaccharide biosynthe sis protein, has been identified as a novel pathogenicity-associated determinant of G. parasuis (10).G. parasuis isolates with capD are highly and moderately virulent (11,12), and CapD expression is upregulated in vitro (13).Therefore, understanding the transcrip tional mechanisms of capD will help to explain the molecular mechanisms of adhesion, colonization, and invasion of G. parasuis.
Two-component systems (TCS) are the dominant form of genetic control in response to environmental stimuli in bacteria (14).In a broad spectrum of bacterial species, the QseBC TCS as a quorum-sensing system is conserved (15).The QseBC controls the expression of some genes linked to metabolism, virulence, and stress response (16).In Edwardsiella tarda and enterohemorrhagic Escherichia coli, the QseBC regulates the transcription of the genes encoding the flagellar proteins in an epinephrine (Epi)-and norepinephrine-dependent manner to control bacterial motility (17,18).In the presence of signal molecules, QseC works as sensor and receives signals, and then transfers the phosphate to the intracellular response regulator QseB.Therefore, QseC inhibitors have been developed as novel antimicrobials (19), and QseC may also be utilized as a potential subunit vaccine candidate (20).In addition, the activation pattern of QseC and the regulatory effects of QseB on virulence genes differ between bacteria (21).The investigation of the relationship between QseBC and the virulence gene capD will help to explain the pathogenic mechanism of G. parasuis.
In the present study, we demonstrated that the response regulator QseB is positively controlled by itself in the current of Epi, which is related to the regulation of CPS synthesis gene capD.As a result, CPS synthesis was inhibited, leading to increased adhesion and reduced biofilm formation of G. parasuis.These results will help us better understand the colonization and pathogenic mechanism of G. parasuis.Reducing stress in pigs is necessary for the prevention and treatment of Glässer's disease, and the development of TCS inhibitors may also be more meaningful in disease control.

Epi affects CPS synthesis, adhesion and biofilm formation in G. parasuis
To determine whether Epi modulates the biosynthesis of CPS, we measured the total carbohydrate content in G. parasuis CF7066 cultured in tryptic soy broth (TSB) unsup plemented or supplemented with 50 µM Epi, respectively, and blanked with the CPS biosynthesis gene deletion mutant CF7066 ΔcapD to accurately quantify the capsules.The relative content of total carbohydrate was assessed to quantitatively compare the difference in CPS formation.As shown in Fig. 1B and C, the addition of Epi resulted in significantly lower amounts of CPS.Subsequently, we observed changes in bacterial adhesion and biofilm formation in response to Epi stimulation, which are directly correlated with capsule polysaccharide content.The adherence ability of G. parasuis CF7066 significantly increased after Epi treatment (Fig. 1D), whereas the biofilm formation ability significantly decreased (Fig. 1E and F), both of which are consistent with a reduction of CPS synthesis.These results showed that the biosynthesis of CPS was considerably reduced under Epi stimulation, indicating that a novel regulatory mecha nism underlies CPS synthesis in G. parasuis.

The response regulator QseB self-regulates its own expression under Epi stimulation
In order to explore the impact of Epi on the transcription and expression of G. parasuis genes, quantitative real-time PCR (qRT-PCR) and Western blot were used to determine mRNA and protein expression, respectively, and we found that the expression of qseB was significantly upregulated in the presence of 50 µM Epi (As shown in Fig. 2E and F).
To identify the full-length mRNA of the qseB operon, a reverse transcription polymer ase chain reaction (RT-PCR) analysis was carried out.Using genomic DNA as a template, six gene-specific primers (Table 2) were used to create the 655 bp P1, 573 bp P2, 358 bp P3, 1,289 bp P4, 1,761 bp P5, and 1,045 bp P6 fragments (Fig. 2A).The corresponding primer sets were applied to run RT-PCR from the first strand of cDNA.As illustrated in Fig. 2B, the genes ygiW, qseB, and qseC were co-transcribed, and a schematic diagram of primer design is shown in Fig. 2A.Following that, we analyzed the ygiW-qseBC operon, the -35 element and the -10 element, and the putative QseB binding sequence (Fig. 2C).Electrophoretic mobility shift assay (EMSA) was performed, and the result showed that QseB could bind to its own promoter in a concentration-dependent manner, the addition of unlabeled probe can compete with the labeled probe, which further demonstrates the binding ability (Fig. 2D), whereas there was no interaction between QseB and the control probe (the promoter DNA of kanamycin-resistance gene).
To investigate the detailed biological function of the adrenaline response regulatory system QseBC in G. parasuis, the deletion mutant ΔqseB was obtained by homologous replacement, and the complementary strain C-qseB was constructed by electro-trans forming the plasmid pSHGB into the ΔqseB.Western blot analysis revealed that the QseB levels in the C-qseB were significantly higher than that in the wild type (WT), whereas the ΔqseB did not express QseB.These findings indicated that the mutant strains had been successfully constructed (Fig. 2G).

QseB can directly interact with the capD gene promoter
The results presented above suggested that QseB may participate in the Epi-medi ated regulation of polysaccharide synthesis.The transcription of CPS genes was then examined by qRT-PCR to determine the possible effects caused by the deletion of qseB.All 12 genes in the CPS gene cluster except lsgB were down-regulated in ΔqseB strain compared to in WT, among which the capD was the most significantly down-regulated gene (Fig. 3A).
We predicted the promoter of G. parasuis CF7066 in BPROM (http://www.softberry.com/berry.phtml?topic=bprom), and found that both the lsgB, which had no transcriptional change in ΔqseB strain, and the capD, which had the largest change, were independently transcribed (Fig. 3B).At the same time, we predicted one putative promoter at 194 bp from the translation start codon of capD gene (Fig. 3E).The transcription of capD gene was then analyzed using RT-PCR.Figure 3C and  D show that the primer combination P2 was unable to amplify the 609 bp DNA fragment covering partial wbgX and capD genes, meanwhile, the primer combination P3 failed to produce the 452 bp DNA fragment containing partial capD and partial wzA genes.These results demonstrated that the capD was transcribed as a single transcription unit.
In addition, the upstream region of capD in G. parasuis was searched for the E. coli consensus sequence for the QseB binding site.In the capD promoter region, one QseB binding sequence (CTTAAN4CTTAG) was discovered in G. parasuis (Fig. 3E).To confirm whether this is a genuine QseB binding site, the promoter fragment was incubated with G. parasuis QseB and then analyzed via EMSA.As shown in Fig. 3F, the capD promoter fragment bound to QseB while competition by an unlabeled DNA probe attenuated the fluorescent signal of the QseB-DNA complex, suggesting that the interaction of QseB with the capD promoter region was specific.These findings proved that QseB can directly bind to the promoter region of capD.The positions of protein-DNA complexes and free DNA probes were shown.Statistical analyses were performed using the two-way analysis of variance.In bar graphs, expression levels were expressed as mean ± standard error of the mean.The statistical significance of the indicated P-values was determined as *P < 0.05, **P < 0.01, and ***P < 0.001.

The capD is related to adherence and biofilm formation
A polysaccharide biosynthesis protein that was connected to G. parasuis pathogenic ity is encoded by the capD gene.In the present study, a capD gene deletion strain was constructed and the relationship between capD and the adherence and biofilm formation ability was analyzed.Additionally, the absence of capD made G. parasuis more likely to adhere to newborn pig tracheal epithelial (NPTr) cells (Fig. 4A), and the ΔcapD essentially lost the ability of biofilm formation (Fig. 4B and C).It demonstrated that the lack of capD gene had a negative impact on biofilm formation, as well as increased adherence.

QseB affects the formation of G. parasuis CPS via interacting with capD
Based on the combination of QseB directly binding to the capD promoter with a putative QseB binding motif located in capD promoter region, we speculated that G. parasuis regulated the expression of capD through QseB in the presence of Epi.In order to test this hypothesis, qRT-PCR was performed to analyze relative expression levels of capD.
The mRNA levels in various strains and conditions were normalized with the concentra tion of the 16S rRNA gene.As shown in Fig. 5A, whether there was Epi, the mRNA levels of capD in ΔqseB were significantly lower than that in the WT and the complemen tary strain (C-qseB).Additionally, following Epi stimulation, the capD transcription was significantly inhibited in WT and C-qseB.Then, we examined changes in CapD expression at the protein levels.In order to prepare immune serum, we first expressed recombined protein rCapD without signal peptide (1-168 aa) in E. coli BL21 and immunized mice.Subsequently, Western blot was used to determine the expression levels of CapD in different strains following Epi treatment.Unsurprisingly, the changing trend of protein levels was consistent with that of the mRNA transcriptional levels (Fig. 5B).The CapD levels in the ΔqseB strain were considerably decreased and were recovered to WT levels in the C-qseB strain.Aside from that, the transcription of capD was significantly down-regulated in both the WT strain and C-qseB strain under Epi stimulation.These results reveal that G. parasuis negatively regulates the expression of CapD in the presence of 50 µM Epi, and the qseB gene is crucial for both Epi stimulation and capD transcription.

DISSCUSSION
G. parasuis is a commensal bacterium in the upper respiratory tract of healthy pigs, and it can invade piglets and cause Glässer's disease under stress conditions (23,24).However, the pathogenic mechanism is still unclear.In the present study, the regulation of capsule polysaccharide expression in G. parasuis was explored for its biosynthesis changes in the presence of Epi.The results revealed that CPS expression was inhibited in the presence of Epi and we further found that the QseBC two-component system negatively regulated the synthesis of CPS in response to Epi.Overall, this study initially explored the pathogenesis of G. parasuis following the perception of Epi.
In some cases, stress can have adverse effects on a variety of immunological mechanisms, alter bacterial pathogenicity, and ultimately affect disease progression (25,26).Epi is the major signaling molecule during stress and its secretion shifts bacteria from a commensal to an invasive pathogenic state (27).Previous studies have shown that stress can result in elevated Epi levels, which can influence bacterial pathogenicity in a variety of ways, such as boosting bacterial growth, improving bacterial adhesion to host tissue, recognizing the host environment, and promoting the expression of virulence factors (28)(29)(30).As the primary quorum-sensing system of bacteria, QseBC can sense and respond to Epi and regulate bacterial growth, motility, biofilm formation, and virulence gene expression (31).In the current investigation, Epi stimulation significantly increased G. parasuis CF7066's ability to adhere to host cells and significantly decreased its capacity to develop biofilms.
The initial phase of the pathogenesis of mucosal microorganisms is commonly associated with colonization, followed by intimate contact with host cells.The reduced amount of capsule polysaccharide leads to the exposure of adhesive molecules, which promotes colonization and uptake.Previous studies have demonstrated that bacterial adherence to host epithelial cells plays an essential role in causing infections (32).The displacement of Kingella kingae capsule could expose a trimeric autotransporter adhesin called Knh and allows Knh to mediate high-strength adherence to the host cell (33).In previous studies, CPS has been shown to reduce adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs (34).In the present study, Epi stimulation dramatically inhibited CPS expression, and both Epi stimulation and capD deletion enhanced the adherence of G. parasuis to host cells, which was as hypothesized.
The quorum-sensing system seemed to be closely related to the bacterial CPS synthesis; much evidence revealed that quorum-sensing system signals such as Fe 3+ and AI-2 regulates CPS synthesis (35).In the present study, G. parsuis CF7066 senses Epi via QseBC TCS, which is consistent with a previous study in which the QseC of G. parsuis MY1902 might sense Epi in the environment and thus regulate bacterial density (36).Additionally, the QseC sensor kinase was a bacterial adrenergic receptor (15).In the present study, quantitative real-time PCR analysis was performed to identify the potential target genes of QseBC regulatory network in G. parasuis CPS biosynthesis loci.Interestingly, in ΔqseB, the genes involved in capsular polysaccharide were significantly down-regulated in comparison to the control, with the exception of the lsgB gene, which encodes a sialyltransferase.It is worth noting that the deletion of qseB resulted in the most pronounced reduction in capD expression.CapD is an integral membrane protein, which catalyzes the first steps in the synthesis of the soluble capsule precursors UDP-D-FucNAc, and the capD mutant had a significantly higher cell surface hydrophobic ity than the wild-type and the complementary strain, resulting in less biofilm production (37) and reduced CPS production (38).In a word, the present study showed that the expression of CPS synthesis gene capD in G. parsuis was regulated via QseBC two-compo nent system.Under the presence of Epi, QseB can bind to the promoter of capD thereby inhibiting its expression.As a result, the synthesis of CPS was significantly decreased, and the biofilm formation was significantly repressed, making it easier for the bacteria to adhere to host epithelial cells.This may be the main mechanism by which G. parasuis transitions from symbiosis to infection and invades piglets under the action of Epi.
In conclusion, we found that in the presence of Epi, the heavily phosphorylated QseB can interact with the capsular polysaccharide synthesis-related gene capD, leading to changes in bacterial adhesion and biofilm formation (Fig. 6).In the present study, we initially investigated the regulatory function of the QseBC two-component system in capsule polysaccharide synthesis, biofilm formation, and adhesion capability under Epi stimulation.Therefore, QseBC two-component system may be a vaccine or drug target candidate for the development of novel antibacterial to treat G. parasuis infections.

Bacterial strains, plasmids, and growth conditions
The bacterial strains used in this study and their sources are listed in Table 1.G. parasuis and its isogenic derivatives were cultured on tryptic soy agar (TSA) or in TSB (Difco Laboratories, Detroit, MI, USA) supplemented with 5% inactivated bovine serum (Tianhang, Hangzhou, Zhejiang, China) and 10 µg/mL nicotinamide adenine dinucleotide (BioFroxx, Einhausen, Germany).E. coli DH5α and BL21 (DE3) were grown in Luria-Bertani (LB) medium (Difco Laboratories, Detroit, MI, USA) at 37°C.Agar was added to the medium at a concentration of 1.5% when a solid medium was desired.When necessary, 50 µg/mL of kanamycin or 20 µg/mL of gentamicin was added to the medium.

Construction of gene deletion and complementary strains
The mutant strains ΔqseB and ΔcapD used in this study were derivatives of G. parasuis serovar 5 strain CF7066.These two mutant strains were constructed by natural trans formation as previously described (41).In brief, the flanking fragments of the target gene and the kanamycin resistance cassette from plasmid pSHK3 were cloned into the pK18mobsacB vector to generate the recombinant plasmids pKBukd and pKDukd, respectively.Each recombinant plasmid (pKBukd or pKDukd) was then introduced into G. parasuis CF7066 via bacterial natural transformation.
All complementary strains were constructed by electro-transforming the shuttle vector pSHG3 containing the target gene.To obtain the complementation plasmid carrying a target gene, the promoter sequence was analyzed using prediction bacte rial promoter (http://linux1.softberry.com).A DNA fragment containing qseB and its promoter was amplified from CF7066 genomic DNA using PCR.The PCR product was then cloned into pSHG3 plasmid to generate pSHG3-qseB.Subsequently, the recombi nant plasmid was introduced into ΔqseB by electroporation.The complementary strains were screened on TSA plates supplemented with 20 µg/mL gentamicin and confirmed by PCR and sequencing.

Quantitative analysis of polysaccharides
Previous research has revealed that CPS of G. parasuis had the main chain -3-β-Glc6P-3-β-Sug-(Fig. 1A) (22).The anthrone assay was used to analyze the synthesis of the CPS as described previously (42) with some modifications.The strains in the logarithmic growth period [optical density at 600 nm (OD 600 ) ≈ 0.6] were harvested by centrifugation at 8,000 × g for 10 min at 4°C, washed with 0.01 M Phosphate buffered saline (PBS) three times and 150 mM Tris-HCl (pH 7.0) once.The bacteria were then resuspended with 2 mL 150 mM Tris-HCl (pH 7.0).Following that, the bacterial solution was mixed with anthrone-sulfuric acid reagent at a volume ratio of 1:3 and heated at 100°C for 15 min.After placing all of the samples on ice, absorbance measurements were taken at 620 nm using a FLUOstar Omega microplate reader, and the amounts of CPS were determined from a standard curve of glucose.CPS concentrations are expressed as micrograms per 10 9 colony forming units (CFU), and the experiments were performed in triplicate.

Adhesion assay
The adhesion assay was used to count the total number of cell-associated (intracellular plus surface-adhered) bacteria, as previously described (43).Bacteria were pelleted, washed twice with PBS, and resuspended in fresh cell culture medium without antibi otics.NPTr cells were cultured as a monolayer in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum.Cells were infected with G. parasuis suspensions at a multiplicity of infection of 20 and 100, respectively, after being cultured to 80%-90% confluency in 24-well plates.The plates were centrifuged at 800 × g for 10 min to promote bacterial contact with the cell monolayer surface and incubated for 2 h at 37°C in 5% CO 2 to allow bacterial adherence.Subsequently, non-attached cells were removed by washing the cell monolayers with PBS five times.After washing, 0.1% Triton X-100 was added to each well to lyse the NPTr cells.The lysates were serially diluted in PBS and plated on TSA plates to count the CFU after 48-h incubation at 37°C.

In vitro biofilm formation assay
A method that has already been described (44) was modified in order to examine the biofilm-formation ability.Briefly, when the cultures had grown to the mid-exponen tial phase, 50 µL of the bacterial solution was transferred to borosilicate glass tubes containing 2 mL of TSB medium, and then incubated vertically with circular agitation (150 rpm) at 37°C for 48 h.To visualize biofilms, the contents of the tubes were sucked with an injector for staining, and the tubes were washed three times with 2 mL of sterile PBS to remove loosely adhering cells.After being air-dried, the tubes were dyed  a WT, wild-type strain; Kan R , kanamycin resistance; Gen R , gentamicin resistance.
with 2 mL of 1% crystal violet solution for 10 min at room temperature.With the use of an injector, the dye solution was extracted from the tubes.The tubes were then thoroughly washed under running tap water, and extra water was removed from the wells as described above.The adherent cells were lysed with 200 µL of 33% (vol/vol) glacial acetic acid, and 100 µL of the solution was transferred to a microtiter plate to obtain the OD of each tube at 630 nm.The findings of each test were averaged after being run three times.

Reverse transcription PCR and quantitative real-time PCR
To obtain reliable results, methods of bacterial total RNA purification (45) and qRT-PCR (46) were applied.In brief, the overnight grown bacterial suspensions were inoculated into fresh medium at a ratio of 1:50 with circular agitation (200 rpm) for 6 h at 37°C until logarithmic growth period (OD 600 ≈ 0.6).Collect the bacteria after mixing twice the volume of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) with the bacterial suspension for 10 min at room temperature.Later, total bacterial RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.The total RNA concentration was quantified by measuring the ratio of OD 260 /OD 280 and then analyzed by gel electrophoresis to determine whether it was intact.After removing the residual genomic DNA with a gDNA wiper (Vazyme Biotech, Nanjing, Jiangsu, China), the cDNA was synthesized with random hexamer primers by using a HiScript III 1st Strand cDNA Synthesis Kit R312 (Vazyme Biotech, Nanjing, Jiangsu, China) according to the manufacturer's instruction.Quantitative real-time PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, Jiangsu, China) on a Bio-Rad CFX96 machine (Bio-Rad Company, Pleasanton, CA, USA).The 16S rRNA gene of G. parasuis from the same sample was used as an endogenous reference and the relative copy number of the target gene's mRNA was calculated using the comparative cycle threshold (2 -ΔΔCT ) method.All data came from at least three independent biological experiments.The primer sequences used for gene expression analysis are listed in Table 2.
RT-PCR was performed to detect the full-length mRNA of qseB and capD operons.Specific primers (Table 2) were designed based on genome sequence information, and genomic DNA and RNA were used as positive and negative controls, respectively.

EMSA
EMSA was carried out in the same way as described in a previous study (47) with some modifications.Briefly, the 194 bp upstream promoter region of the capD gene and qseB gene was amplified, respectively, by PCR with labeled primers to generate the Cy5.5-capD and Cy5.5-qseB promoter fragment from CF7066-WT genomic DNA.The amplified fragment was purified using the Gel Extraction Kit (Omega Bio-Tek Inc., Norcross, GA, USA).The purified recombinant protein QseB (rQseB) was dialyzed against binding buffer (50 mM Tris, 10 mM MgCl 2 , 0.1 mM dithiothreitol (DTT), pH 7.0).Then, 30 ng of the labeled probe and increasing amounts of rQseB were incubated for 30 min at room temperature in binding buffer; 20 mM lithium potassium acetyl phosphate (Sigma-Aldrich, Steinheim, Germany) was added to the reaction system.Unlabeled PCR products (600 ng) were used as competitive probes in competitive experiments.After adding 10% (vol/vol) volume of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) to the reaction mixtures, electrophoresis was performed on a 6% polyacrylamide gel in 0.5× Tris-Borate-Ethylenediaminetetraacetic acid (TBE) buffer (45 mM Tris, 45 mM boric acid, 0.5 mM EDTA, pH 8.0) at 4°C and 100 V for 1 h, and the gels were exposed to an Amersham Typhoon 5 (GE HealthCare, CA, USA).

Recombinant protein expression and purification
The qseB and 1,437 bp sequence omitting the signal peptide sequence of capD were amplified by PCR using the rQseBF/R and rCapDF/R primers (Table 2) and CF7066  genomic DNA as the template, respectively.The PCR products were gel purified using the Gel Extraction Kit (Omega Bio-Tek Inc., Norcross, GA, USA).Then, the amplified fragments were digested with restriction enzymes BamHI/XhoI (New England Biolabs, Ipswich, MA, USA) or BamHI/HindIII (New England Biolabs, Ipswich, MA, USA), and ligated to the similarly digested pET28a.The recombinant plasmids were designated as pET-qseB and pET-capD and were introduced into E. coli BL21 (DE3) (Transgen Biotech, Beijing, China).E. coli BL21 (DE3) cells containing recombinant plasmid were respectively cultured in LB medium in a shaking incubator at 37°C.When the cultures were grown to the OD 600 values of 0.4-0.5, isopropyl-β-d-thiogalactoside was then added at a final concentration of 0.8 mM to induce expression at 37°C for 5 h.The bacteria were harvested by centri fugation at 12,000 × g for 5 min and washed with PBS three times.The harvested cells were suspended in binding buffer and broken by passing through a pressure cell disruptor three times.The rQseB was purified as previously described (48) by Nickel nitrilotriacetate (Ni-NTA) resin affinity chromatography (Qiagen, Hilden, Germany).However, the CapD protein was expressed as inclusion body, from which correctly folded protein can be easily extracted using nondenaturing solvents (49).The purified protein was confirmed by Western blot with an anti-His tag antibody (ABclonal, Wuhan, Hubei, China).

Western blot analysis
Antisera against QseB and CapD proteins were raised in mice using recombinant proteins.The BALB/c mice (6 weeks old) were immunized with proteins via subcutaneous injection.A protein dose of 25 µg in a total volume of 200 µL PBS was given with Freund's Incomplete Adjuvant in 1:1 (vol/vol) emulsion.Secondary immunization was administered 2 weeks later.One week later, blood was collected from mice with their tails severed.After that, serum was extracted from the blood and tested by indirect enzymelinked immunosorbent assay.Western blots were performed to detect changes in QseB and CapD levels in extracts from wild-type, mutants of G. parasuis and Epi conditions.A total of 10 µg protein, as determined by the bicinchoninic acid assay (Biosharp, Hefei, Anhui, China), was loaded per lane and proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Proteins were then transferred to the polyvinylidene difluoride membrane.After transfer, the polyvinylidene difluoride membrane was incubated with anti-QseB or anti-CapD antiserum at 4°C for 12 h, and Anti-GAPDH Mouse Monoclonal Antibody was from Proteintech Group (Wuhan, Hubei, China).Horseradish peroxidase (HRP) Goat Anti-Mouse IgG (H + L) (Abclonal, Wuhan, Hubei, China) was used as a secondary antibody.The results were visualized with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA).

Statistical analyses
The comparison of several series was completed using one-way analysis of variance (ANOVA) or two-way ANOVA.Student's t-test was used to determine the differences between groups.Value P < 0.05 was regarded as statistically significant.

FIG 1 FIG 2
FIG 1 Examination of G. parasuis carbohydrate contents, adherence ability, and biofilm formation under Epi stimulation.(A) Structure of the capsular polysaccharide in G. parasuis serovar 5 (22).Sug, 2,4-diacetamido-2,4,6-trideoxy-ᴅ-galactopyranose; R, acetyl (Ac) or glycolyl (Gc); italic, non-stoichiometric components.(B) G. parasuis was cultured in TSB unsupplemented or supplemented with 50 µM Epi, and total carbohydrate content was determined and blanked with the capD deletion mutant ΔcapD to accurately quantify the CPS (n = 3).(C) Epi stimulation dramatically inhibits CPS synthesis (n = 3).(D) The bacteria in logarithmic growth phase cultured in TSB unsupplemented or supplemented with 50 µM Epi were selected to interact with newborn pig tracheal epithelial cells at multiplicity of infection 20 and 100 for 2 h.The data represent the number of bacteria that adhered to the cells in each well of a 24-well plate (n = 3).(E)Biofilms of wild type cultured in the absence (control) or presence of 50 µM Epi at 48 h were stained with crystal violet.(F) Quantification of biofilm production (n = 3).Statistical analyses were performed using the one-way analysis of variance.In bar graphs, expression levels were expressed as mean ± standard error of the mean.The statistical significance of the indicated P-values was determined as *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG 2 ( 5 FIG 3 G
FIG2 (Continued)    region.The underscore sequences are predicted -35 and -10, inside the rectangle is the predicted QseB binding site.(D) Labeled qseB promoter DNA sequences (30 ng) were incubated with different amounts of purified QseB protein (0 µM, 0.3 µM, 0.6 µM, 0.9 µM, and 1.2 µM) in the presence of acetyl phosphate.About 200-fold higher amount of unlabeled qseB promoter DNA sequences were used as specific competitors.The positions of protein-DNA complexes and free DNA probes were shown.(E) Western blot analysis to detect effect of Epi on QseB protein expression.(F) Quantitative real-time PCR analysis of the relative changes of qseB mRNA levels in the presence of Epi (n = 3).(G) Confirmation of the deletion mutant ΔqseB and the complementary strain C-qseB using Western blot.The statistical significance of the indicated P-values was determined as *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG 4 G
FIG 4 G. parasuis capD was related to capsular polysaccharide synthesis, adhesion, and biofilm formation ability.(A) NPTr cells were inoculated with the indicated strains at multiplicity of infection 20 or 100, culture plates were incubated for 2 h.The data represent the number of bacteria that adhered to the cells in each well of a 24-well plate (n = 3).(B) Biofilms of WT and ΔcapD at 48 h were stained with crystal violet.(C) Quantification of biofilm production.Statistical analyses were performed using the one-way analysis of variance.In bar graphs, expression levels were expressed as mean ± standard error of the mean (n = 3).The statistical significance of the indicated P-values was determined as *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG 5 G
FIG 5 G. parasuis QseB negatively regulates capD in the presence of 50 µM Epi.(A) The expression levels of capD in strains WT, ΔqseB, and C-qseB with 50 µM Epi stimulation were analyzed by qRT-PCR (n = 3).(B) CapD levels in strains WT, ΔqseB, and C-qseB were analyzed by Western blot using polyclonal antibodies raised against CapD.Protein extracts (100 µg) were obtained from bacteria in logarithmic growth phase cultivated in TSB unsupplemented or supplemented with 50 µM Epi.In bar graphs, expression levels were expressed as mean ± standard error of the mean.The statistical significance of the indicated P-values was determined as *P < 0.05, **P < 0.01, ***P < 0.001, and P > 0.05 ns (not significant).

pKBukd A 1 ,
903 bp overlap fragment containing Kan R , and the upstream and downstream fragment sequences of the qseB gene in pK18mobsacB, Kan R This study pKDukd A 1,961 bp overlap fragment containing Kan R , and the upstream and downstream fragment sequences of the capD gene in pK18mobsacB, Kan R This study pSHG3-qseB pSHG3 vector containing deleted qseB gene and corresponding promoter for complementation This study pET-28a Expression vector, Kan R Novagen pET-QseB pET-28a vector containing QseB encoding sequence This study pET-CapD pET-28a vector containing CapD encoding sequence This study of pET28a::qseB vector.P-qseB-R CCGctcgagTTTAAGCAACTTCATCGTTTTTTC P-capD-F CGCggatccCGTTCCCATGATTATTTGC Used for construction of pET30a::capD vector, truncated protein (168-646 aa).P-capD-R CCCaagcttGTTTATCTACTTGCATCCGCC m-16S rRNA-F ACACTGGAACTGAGACAC Used for relative qRT-PCR to check the mRNA transcription levels of corresponding genes.m-16S rRNA-

a
The restriction sites in the primers are marked in lowercase; letters are underlined to indicate the uptake signal sequences (USS) of G. parasuis.

TABLE 1
Bacterial strains and plasmids used in this study a

TABLE 2
Primers used in this study a

TABLE 2
Primers used in this study a (Continued)